A Technique to Generate Monoclonal Antibodies from Antigen-Specific B Cells

프로토콜

1. Production of mAbs

1. The day before the transfection, seed 15,000 Human embryonic kidney 293A (HEK 293A) cells in 200 µL of Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS per well in 96-well plates. Incubate overnight in a CO₂ incubator (5% CO₂) at 37 °C.
2. Cotransfect 293A cells in DMEM medium containing 10% FBS using a linear polyethylenimine derivative as transfection reagent.
   NOTE: Perform the transfection in triplicates. The following protocol is for one well of a flat bottom 96-well plate.
   1. Dilute 0.5 µL of DNA transfection reagent into 10 µL of 150 mM NaCl. Dilute 0.125 µg of variable heavy chain (vH) and 0.125 µg of variable light chain (vL) expressing vectors into 10 µL of 150 mM NaCl. Vortex each dilution for 10 s.
   2. Add 10 µL diluted DNA transfection reagent into the 10 µL DNA solution. Vortex 15 s and incubate for 15 min at room temperature. Add the 20 µL mix drop-wise on 293A cells, and mix by gently swirling the plate.
3. Replace the medium 16 h after transfection and culture cells for 5 days in serum-free medium.
   NOTE: Use serum-free medium to avoid serum immunoglobulins (Igs) contaminating the produced antibodies.
4. Eliminate cells and debris by centrifugation at 460 x g for 5 min.
5. Harvest supernatants by aspiration with a multi-channel pipette and transfer them into a V-bottom 96-well plate.
6. Coat relevant Ag overnight at 4 °C in 100 µL per well of reconstituted enzyme-linked immunosorbent assay (ELISA)/ enzyme-linked immunosorbent spot (ELISPOT) coating buffer 1x at a final concentration of 2 µg/mL in 96-well ELISA plates.
7. Block wells with 10% FBS DMEM medium for 2 h at 37 °C.
8. Add 50 µL of supernatants of transfected 293A cells from step 1.5, and incubate for 2 h at RT.
9. Add 100 µL of an anti-human Immunoglobulin G antibody (IgG Ab) conjugated to horseradish peroxidase (HRP) enzyme at 1 µg/mL and incubate for 1 h at room temperature. Add 50 µL chromogenic substrate (3,3',5,5' tetramethylbenzidine, TMB), and incubate for 20 min.
10. Read optical densities at 450 nm on a spectrophotometer.
    NOTE: The specific mAbs revealed by ELISA assay can be further produced at large scale and purified on protein A column presenting affinity for human IgG.

Access restricted. Please log in or start a trial to view this content.

자료

Name	Company	Catalog Number	Comments
HEK 293A cell line	Thermo Fisher scientific	R70507
DMEM (1X) Dulbecco's Modified Eagle Medium	Gibco by life technologies	21969-035	(+) 4,5g/L D-Glucose 0,11g/L Sodium Pyruvate (-) L-Glutmine
Nutridoma-SP	Roche	11011375001	100X Conc
Fetal Bovine serum (FBS)	Dominique Dutscher	S1810-500
Jet PEI DNA transfection reagent	PolyPlus	101-40
Flat bottom96-well plate	Falcon	353072
V-bottom 96-well plate	Nunc/Thermofisher	55142